Progress culminating in the last year includes preparation of large crystals of cytochrome P450cam now to be tested by x-ray for appropriateness in determining the crystal's three dimensional structure. Protein has been prepared in apo form and reconstituted with iron and cobalt hemes to provide isotope replacement suitable for physical probes and physical characterization of heme pockets in the environment. The stable isotope, 33 sulfur, in the maximum available abundance has been used to grow cells. Pure enzymes have been subjected to EPR and NMR analysis to characterize the axial iron ligand. Optical spectroscopy methods have been used to extend our understanding of effector substrate on heme pocket modulation in aqueous and cosolvent environments. Amino acid residues have been modified and the effect on iron spin state and substrate binding has been elucidated. A second millimolar, as opposed to a micromolar, catalytic site has been observed for substrate. These advances perimt physical structure and functional analysis to elucidate monoxygenases of the first and second redox reaction and the effects of carbon and oxygen substrates. These studies should result in better understanding of the three dimensional structure, the modulation and regulation of energy and substrate flow, and the caralytic role of the heme protein. Our current research group contains the skilled physical, biochemical, and genetic scientists to move into the next stage of exploring the primary reactions permitted with multisubstrate, multienzyme redox catalysis.